Rhizocarpon geographicum. We sampled R. geographicum in French habitats exposed to oil spills, and we combined nine culturing methods with 16S rRNA sequencing to capture the greatest bacterial diversity. A deep functional analysis of the lichen-associated bacterial collection showed the presence of a set of bacterial strains resistant to a wide range of antibiotics and displaying tolerance to Persistent Organic Pollutants (POPs). Our study is a starting point to explore the ecological features of the lichen microbiota.As rock inhabitants, lichens are exposed to extreme and fluctuating abiotic conditions associated with poor sources of nutriments. These extreme conditions confer to lichens the unique ability to develop protective mechanisms. Consequently, lichen-associated microbes disclose highly versatile lifestyles and ecological plasticity, enabling them to withstand extreme environments. Because of their ability to grow in poor and extreme habitats, bacteria associated with lichens can tolerate a wide range of pollutants, and they are known to produce antimicrobial compounds. In addition, lichen-associated bacteria have been described to harbor ecological functions crucial for the evolution of the lichen holobiont. Nevertheless, the ecological features of lichen-associated microbes are still underestimated. To explore the untapped ecological diversity of lichen-associated bacteria, we adopted a novel culturomic approach on the crustose lichen  The term symbiosis was first introduced by Albert Bernhard Frank in 1876 to describe the mutualistic association between a fungal partner and a photobiont leading to a lichen symbiotic organism . DespiteSince the beginning of the 20th Century, lichen-associated bacteria were described to harbor ecological functions crucial for the maintenance of the lichen symbiotic system ,12,13,14In order to dissect the ecological functions behind the microbe–lichen interaction, the culturable and the unculturable microbiota need to be deeply explored. Studies on the lichen microbiota are mostly based on molecular fingerprints ,19,20, mRhizocarpon geographicum, a crustose lichen pioneer of exposed rock surfaces and the first terrestrial substrate available for living organisms on Earth [R. geographicum in French zones strongly affected by hostile environmental conditions . Moreover, three of the sampling locations had a history of oil spills. As a previous study revealed that the interaction of mutagenic compounds in natural environments is correlated with the development of antibiotic resistance genes in bacteria [R. geographicum microbiota. We pointed out the presence of a set of bacterial strains resistant to a wide range of antibiotics and displaying tolerance to two POPs: Methyl Tert-Butyl Ether (MTBE) and Perfluorooctanoic Acid (PFAO). Our study stresses the need to consider lichens as both sources of important ecological functions and reservoirs of biotechnologically relevant bacterial species.Here, we adopted culturomics on on Earth ,29. We sbacteria , we inveR. geographicum samples were collected in January 2021, under specific municipality authorizations, in 6 sites situated in France (R. geographicum population), lichen samples were collected randomly on four rocks (n France a on: (i)n France b–d. Threur rocks , and rocR. geographicum was scraped and mixed with 100 mL of filtrated seawater collected from the site of sampling. The suspension was centrifuged for 10 min at 5000 rpm at 4 °C. The supernatant was recovered and sterilized by filtration through 0.22 µm pore-size Millipore® membranes. The second type of organic matrix was performed on a macroalgal filtrate. For this, 155 g of Ascophyllum nodosum and 95 g of Laminaria digitata collected at la Pointe de Crozon  were rinsed with seawater. These algae co-habitate in the ecosystem with R. geographicum and are part of the lichen’s natural habitat. Macroalgae were roughly cut before being homogenized with a clean kitchen blender then bag-mixed with a Stomacher. From the concentrated solutions, 400 mL was recovered and filled with 600 mL of seawater collected at la Pointe de Crozon, previously sterilized by filtration. Once crushed, the algal pastes were passed through a stamen. Filtrates were then centrifuged for 10 min at 5000 rpm at ambient temperature. Filtrates were then filtered through 0.22 µm pore-size Millipore® membranes to guarantee sterility. The third organic matrix consisted of a microalgal filtrate. Specifically, Coccomyxa viridis, a microalgal associated with R. geographicum, collected on the Atlantic coastal area in Crozon, was previously isolated on International Streptomyces Project 2 medium , yeast extract 4 g·L−1 , malt extract 10 g·L−1 , agar 15 g·L−1 ). The total mass present in one Petri dish of C. viridis was added into 50 mL of NaCl 0.85%, then transferred into 500 mL of ISP2 liquid medium. The culture was maintained under agitation (120 rpm) for 2 weeks. After the growing time, 500 mL of filtered sea water was added into the C. viridis suspension and sterilized by filtration as described above.Three types of matrices were prepared as organic nutrient sources and were supplemented into the media. For the first type of matrix, we developed a lichen filtrate. For this, 50 mg of −1 of chloramphenicol, 300 mg·L−1 of streptomycin, and 100 mg·L−1 of penicillin G to isolate antibiotic-resistant strains. Detailed protocols for media preparation are listed in the The lichen-, macroalgal-, and microalgal-based matrices were then used to develop culturing media . The med® tubes containing 5 mL of sterilized distilled water. The fresh mass of each lichen used for isolation is listed in −1 to 10−3) were directly plated on the 9 media , described in the R. geographicum population were pooled, and 100 µL of each lichen suspension was added to the enrichment broth. The Erlenmeyer flasks were sealed with a cotton and aluminum cap and maintained under agitation (100 rpm) at 15 °C for 4 weeks. Once a week, the cap was aseptically removed for 15 min to aerate the cultures. After 4 weeks of incubation, dilutions from the enrichment solutions were plated in triplicate on the 9 media , colonies showing distinct morphologies were purified on the medium of origin and stored at −20 °C in 30% of glycerol. Pure colonies were placed into 96-well plates containing a DNA-stabilizing buffer composed of 10 mM TRIS, pH 8.0, 0.1M EDTA, pH 8.0, and 0.5% SDS. Plates were stored at −20 °C prior to DNA extraction, which was performed with the protocol described in Vingataramin and Frost (2015) . Bacteriexp) and stationary (sta) phase. After incubation, 10 µL of each strain at the exp or sta phase was inoculated into sterile 96-well plates containing 90 µL of TSB and the appropriate antibiotic concentration  at a final concentration of 2 g·L−1. Controls consisted of strains inoculated on TSB only. POP screening was performed on 96-well plates containing a volume of 200 µL of TSB supplemented with the POP. Each well was inoculated with one pure bacterial colony. The inoculated 96-well plates were incubated for 7 days at room temperature, then the ODs were measured at λ = 620 nm with the microplate reader Multiskan™ FC, Thermo Scientific™. The experiment was independently repeated two times , and each block was constituted by three replicates. We estimated the POP tolerance coefficient by dividing the OD of each strain growing in the presence of the given POP by the OD of the strain growing in the absence of the POP. Coefficient values were integrated into a generalized linear-mixed model that was run by using the lmer function implemented in the lsmeans and lme4 R packages [A sub-set of 394 bacterial strains belonging to unique 16S clusters and showing a suitable growth rate when re-cultured on the TSA medium after storage were tested for their ability to tolerate and grow in the presence of Persistent Organic Pollutants (POPs). For this, the 394 strains were grown for 3 days on TSA and then inoculated into TSB supplemented either with Perfluorooctanoic Acid (PFOA) at a final concentration of 20 mg·Lpackages ,33. The R. geographicum was sampled in six highly diversified maritime and terrestrial habitats located in France  showed that the CARO-RG-8B-R23-01 strain belongs to the Microbacterium lacticum species; on the other hand, CARO-RG-8B-R24-01 belongs to a potential new species of the Sphingomonas genus , were significant on 5 of the 12 antibiotics tested  Pseudomonas antartica for the TREG-RG-20F-10-E-5-01, TREG-RG-20F-10-E-6-0, MONT-RG-20F-10-E-7-02, and MONT-RG-20F-R14-5 strains; (iii) Sphingomonas glacialis, Sphingomonas panacis, and Sphingomonas ginsenosidivorax for the TREG-RG-20F-R18-01, BAUL-RG-20F-R05-02 and CROZ-RG-20F-R02-07 strains, respectively   . Interesntartica  and carrntartica , showed ntartica To better understand the genetic bases of the antibiotic resistance activity of the nine strains, we investigated the presence of Antimicrobial Resistance (AMR) proteins by using AMRFinder Plus , enablin2 = 483.04, p = 0.001425) on the MTBE tolerance and on the PFOA tolerance  by suggesting a high variability among the strains in response to both POPs tested  and the possible connection with antibiotic resistance activity, Perfluorooctanoic Acid (PFOA)—a perfluoroalkyl substance found in aqueous film-forming foams—and Methyl Tert-Butyl Ether (MTBE)—a petrol additive—were used as the sources of POPs during the bacterial growth of 394 strains . The 394s tested . Regresslerances .Frondihabitans, an actinobacterium known to colonize lichens; (ii) Arthrobacter, a ubiquitous bacterial species colonizing several substrates; (iii) Paenibacillus, a lichen-associated bacterium; (iv) Amantichitinum, a soil bacterium; (v) Deinococcus alpinitundrae, isolated previously from alpine environments and known to be resistant to ionizing radiation; (vi) five strains belonging to the Pseudomonas and Sphingomonas genera.For the MTBE, within the 394 strains, 237 were able to tolerate the POP (tolerance coefficient ≥ 1) and 42 strains had a tolerance coefficient ≥ 1.5. The fifteen strains (3.8% of the tested bacteria) showing the highest level of tolerance (coefficient ≥ 2) were recovered from the Carolles (CARO), Crozon (CROZ), and Trégastel (TREG) populations , Table 2Pseudomonas, Bacillus, Sphingomonas, and Paenibacillus genera, suggesting a recurrent ability of these bacterial species to degrade a wide range of POPs. On the other hand, we also found that some of the most-tolerant strains were affiliated with the genera Methylobacterium, Erythrobacter, Micrococcus, and Frigoribacterium. In our study, POP tolerance was not correlated with antimicrobial activity, as described previously [For the PFOA, 216 of the 394 tested strains showed a tolerance coefficient ≥ 1 and 39 strains a coefficient ≥ 1.5. Thirteen strains (3.2% of the tested bacteria) were highly tolerant to the PFOA, and as for the MTBE, most of them were isolated from the Carolles, Crozon, and Trégastel lichen populations . This reeviously . On the R. geographicum. Most of the previous culture-dependent studies on lichens only used classic media, allowing isolating a small fraction of lichen-associated microbes [R. geographicum. Firstly, Bjelland et al. [R. geographicum without combining culturing methods. Secondly, a recent study from Miral et al. [R. geographicum by using classical microbiological media. Miral and co-authors did not use antifungal compounds for bacterial isolation; therefore, they isolated a low bacterial diversity. For instance, most of the isolated bacteria belonged to the Paenibacillus etheri species. Our study is the first deep ecological investigation of bacterial communities associated with R. geographicum, a lichen undoubtedly difficult to sample from rock surfaces, but adapted to extreme environmental conditions. To our knowledge, only a few works have used original media enriched with lichen extract in order to isolate microbes from other lichen species [R. geographicum environment allowed isolating bacterial species not identified before on conventional media. Our results claim that host-specific nutrients play a substantial role in isolating rare species. Developing culturing methods on these nutrients is the missing key to build diversified microbial collections from lichens with an undiscussed importance for eco-evo studies on holobionts such as lichens. Indeed, among the 699 single clusters amplified and characterized with the 16S marker gene, 19.9% of the bacteria were identified as uncultured, including an unclassified strain and three strains without any matches when blasted on GenBank. Among the 11 whole-genome-sequenced strains, nine were putative novel species. Moreover, the long incubation time allowed recovering strains belonging to the Frankiales order [R. geographicum microbial diversity, Bjelland et al. in 2011 [R. geographicum bacterial communities consisted mainly of Acidobacteria, Proteobacteria (α- and β-Proteobacteria), and Chloroflexi classes, with a poor discrimination at the order level due to a lack of metabarcoding resolution. In agreement with previous metabarcoding studies, by culturomics, at the bacterial class level, we also found that α-Proteobacteria and β-Proteobacteria were the predominant bacterial classes of the R. geographicum microbiota. In addition, we isolated a large amount of Actinomycota. Furthermore, in our study, we deeply characterized the isolated bacteria at the family and species level, and we succeeded in isolating rare species previously known to be adapted to extreme environments  against various multi-resistant Staphyloccocus aureus strains [R. geographicum was sampled in littorals with a history of oil spills, the resistome (the complex of AMR genes) might have emerged in lichen-associated bacteria in response to pollutants to help the bacteria be resilient in contaminated habitats [Pseudomonas was dominant in PAH-contaminated sites and that hydrocarbon degradation ability and antibiotic resistance were strongly correlated in these bacteria [AMR gene analyses on the 11 genomes showed resistance mechanisms for antibiotics, such as classes of β-lactamases and Resistance–Nodulation–Division (RND) efflux pumps for strains identified as potential novel species. The antibiotic resistance pathways found in these strains are common in Gram-negative bacteria  and well species ,71. For  species ,73,74. Te strain . Taken te strain ; these c strains . The pre strains . Further strains ,79, a gr strains ,80 as ex strains ,81. Takihabitats ,83. In abacteria . In addibacteria ,86.Our results demonstrate that, on average, 60% for MTBE and 54% for PFOA of the strains tested for POP resistance were able to grow and tolerate the POPs (tolerance coefficient ≥ 1). However, unlike what was described before, resistance to the POPs was not correlated with antibiotic resistance, when considering the most-tolerant strains (tolerance coefficient ≥ 1.5 and 2). Interestingly, strains with the highest tolerance coefficient were isolated from lichens that experienced a history of oil spills. This observation indicates that pollution and environmental constraints can select from bacterial lineages with the ability to tolerate and, probably, metabolize pollutants. Therefore, mass isolation and characterization of strains from these particular habitats can help in identifying novel microbial candidates for biotechnological purposes. In this context, culturomics needs to be developed for selecting highly pollutants-degrading strains. It is worth noticing that some POP-tolerant strains were not recovered from habitats with an oil spill history. Regarding this, as lichens are known to be extreme habitats for microbes, resistance and tolerance mechanisms might evolve without a precise pollutant selective pressure. As PAHs and the POPs tested in our study are organic substances characterized by their persistence, toxicity, mobility for long distances, and their bio-accumulative nature , the bac


Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that lung injuries caused by PS-NPs were mediated via redox imbalance, which was verified by reactive oxygen species (ROS) staining. Additionally, we obtained ten key transcription factors (TFs) governing differentially expressed genes (DEGs), nine of which were involved in the regulation of oxidative stress. An oxidative stress-associated TF-mRNA regulatory network was constructed on account of the findings above. Further joint analysis with animal experiment data from the GEO database identified a crucial oxidative stress-related molecule, TNFRSF12A. qRT-PCR was performed to confirm the results of RNA-seq. (4) Conclusions: Our study indicates the potential role of oxidative stress in the mechanism of nanoplastics-induced lung injuries, with several key genes being promising targets to analyze in future investigations.(1) Background: Increasing evidence reveals that airborne plastic particles will continue to degrade into nanoplastics which are then inhaled by humans, causing injury to the respiratory system with controversial molecular mechanisms. (2) Methods: We used polystyrene nanoplastics (PS-NPs) as the representative pollutants to explore the inhalation toxicology of nanoplastics and identified the potential mechanism through high-throughput sequencing. (3) Results: PS-NPs inhibited cell viability in a dose-dependent manner and 0 μg/cm Plastic materials are widely used all over the world because of their durability, lightness, versatility and flame retardancy ,2. It isMicroplastics are typically considered to have a dimension between 1 μm and 1000 μm, while nanoplastics are considered to be up to 100 nm in size . To dateLimited by conventional detection technology, nanoplastics are difficult to characterize and most plastic particles reported in the environment are 50 μm to 5 mm in size. But recent research has confirmed that microplastics in the environment will not stop disintegrating into nanoplastics ,21,22. MPolystyrene, as one of the main components of plastic, has broad applications in electronics, packing products, insulating foams, etc. . FurtherDespite some recent data suggesting a potential association between airborne nanoplastic exposure and pulmonary damage, further research is still needed to fully understand the underlying molecular events. In this study, we performed transcriptome sequencing (RNA-Seq) on human lung epithelial cells (BEAS-2B cells) exposed to low and high concentrations of PS-NPs (40 nm in diameters) to explore the potential mechanisms. We tested two types of human lung epithelial cells, including HPAEpiC and BEAS-2B cells, and then combined our findings with data from animal experiments to identify potential key genes. Our research aimed to offer a fresh viewpoint on the investigation of respiratory toxicity caused by nanoplastics.BEAS-2B cell line was supplied by the American Type Culture Collection  and HPAEpiC cell line was from ScienCell Research Laboratories . BEAS-2B cells and HPAEpiC cells were grown in DMEM and an RPMI-1640 medium, respectively, containing 10% fetal bovine serum  and 1% penicillin-streptomycin antibiotics . The stock of unmodified polystyrene nanoparticles (PS-NPs) with a size of 40 nm was supplied by Shanghai Huge Biotechnology Corporation . The characterization of the PS-NPs was evaluated as previously described . Before 2, which were equivalent to 0, 8, 16, 24, 32, 48, 64, 80, 96, 112 and 128 μg/mL with 100 μL culture medium per well in the 96-well plate. After overnight incubation, the CCK-8 assay  was performed to determine cell viability under the guidance of the manufacturer’s protocol.Cells were grown in 96-well plates to 70% confluence and then treated with the PS-NP suspension. A total of eleven groups of concentration gradients were set, including 0, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35 and 40 μg/cm2O2 diluted with a serum-free medium to 100 μmol/L was used as the positive control. DCFH-DA was diluted with a serum-free medium to a final concentration of 10 μmol/L. The cell culture medium was then removed, and an appropriate volume of diluted DCFH-DA and H2O2 was added to fully cover the cells. Cells were incubated at 37 °C for 30 min. Intracellular ROS (DCFH-DA) was detected using fluorescence microscopy with excitation at 488 nm and emission at 525 nm.The intracellular ROS levels were measured using DCFH-DA  with a fluorescence microscope according to the instructions of the manufacturer. H2, 7.5 μg/cm2 and 30 μg/cm2) were selected for the analysis of the differentially expressed genes (DEGs) and three parallels were set in each group. Nano-Photometer® spectrophotometer  and Qubit®3.0 Fluorometer  were used to detect the purity and concentration of the total RNA. The RNA integrity was checked using Agilent 2100 RNA nano 6000 assay kit . After passing quality control, RNA samples were sequenced using an Illumina system with PE150 as the sequencing strategy. The Fragments per Kilobase per Million Mapped Fragments (FPKM) method was used to calculate the gene expression. The DESeq2 method was used to identify DEGs with the criteria of fold change and FDR . The sequencing depth was 6 Gb per sample.Three concentrations of PS-NP treatment groups  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed. The GO terminology was described in three parts: biological processes (BPs), cellular components (CCs) and molecular functions (MFs). The KEGG pathway analysis was conducted to enrich the significant signaling pathways.http://david.abcc.ncifcrf.gov/, accessed on 8 July 2022) was used to obtain the TFs regulating the DEGs, and 172 TFs were subsequently identified with the criteria of p-value < 0.05. After intersecting 172 TFs with the DEGs, we captured ten key TFs that were differentially expressed with the treatment of PS-NPs in BEAS-2B cells. To collect the TFs and DEGs involved in oxidative stress, we searched the keywords “oxidative stress” in the GeneCards database  to generate the gene set related to oxidative stress, including 9438 candidate genes  which is a public collection of functional genomics data encompassing significant high-throughput sequencing experimental data. The GSE13535 dataset includes a rat lung gene expression during an acute pulmonary embolism. Rats were intravenously injected with 2 concentrations of 25-micron polystyrene  and 0.15 mL/100 g weight of 0.01% Tween20 as a “vehicle” control. Rats were sacrificed at either 2 h or 18 h after treatment. Finally, we chose the 18 h group for analysis by GEO2R considering that far more DEGs were available in the longer treatment group.The microarray dataset GSE13535 was obtained from Gene Expression Omnibus  was used to extract total RNA. Hiscript II Q RT SuperMix  was used to perform a reverse transcriptase reaction. Moreover, real-time PCR reactions were performed using SYBR Green PCR Master Mix . GAPDH was selected as the internal control and ΔΔCt methods were used for normalization. The Ct values of the target genes were normalized to the control gene, and the mRNA expression was presented as the relative fold change. qRT-PCR experiments were performed in triplicates. The sequences of all the primers for qPCR used in the study are listed in p-value < 0.05 was considered statistically significant.Graphpad prism 9.0  was used for statistical analysis. The experiments were conducted in triplicate and repeated 3 times independently. All the data were expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was performed for statistical difference analysis in the study. 2 PS-NPs. It also revealed that BEAS-2B cells were susceptible to the damage caused by PS-NPs, even at a concentration as low as 10 μg/cm2. In addition, the cell viability dropped to approximately 70% when the treated concentration reached 30 μg/cm2. For the purpose of capturing the responsive genes induced by PS-NPs in BEAS-2B cells at low-concentration treatment and in a relatively high-concentration group, we selected 7.5 μg/cm2 (low-dose group) and 30 μg/cm2 (high-dose group) as exposure concentrations and 0 μg/cm2 as the control group for the subsequent RNA-seq.BEAS-2B cells were treated with different concentrations of PS-NPs for 24 h and then the cell viability was measured by CCK-8 assay. As illustrated in 2 (control group), 7.5 μg/cm2 (low-dose group) and 30 μg/cm2 (high-dose group) groups. Compared with the control group, 2531 genes were significantly altered in the 7.5 μg/cm2 group, with 1231 genes up-regulated and 1300 genes down-regulated  and six down-regulated genes . BEAS-2B cells and HPAEpiC cells were treated with PS-NPs with concentrations of 7.5 μg/cmus study . FortunaNanoplastics have become predominant contaminants nowadays, which has raised great concern about their toxicity . Due to 2O2 and oxidative stress, was used in this study to verify the results of the KEGG and GO analyses. We found that ROS levels remarkably increased in BEAS-2B and HPAEpiC cells after PS-NP exposure for 24 h. Since previous evidence showed that oxidative stress induced by the excessive accumulation of ROS acted as the critical adverse key event to initiate downstream signaling cascades and ultimately caused cytotoxicity [Here we had shown that PS-NPs lead to dose-dependent cytotoxicity in BEAS-2B cells. In order to more accurately reveal the dynamic changes of genes in BEAS-2B cells induced by PS-NP exposure, we chose three groups  for transcriptome sequencing. A total of 1812 genes were changed with a consistent trend in the two treated groups. Notably, approximately 700 genes had significant changes in low- but not high-dose-treated cells, and these genes may play crucial roles at sublethal stages. Sublethal damage caused by low-concentration treatments of either the PS-NPs in this study or other irritants is often reversible, and after the elimination of the irritant environment, the cells may be able to return to their normal state. Therefore, studying the genes that change in the low-concentration group alone could also guide the investigation of the potential repair mechanism of the injury, which represents a significant step forward in the prevention and treatment of diseases. Our present study mainly focused on those pivotal genes that play a role in both the onset and late stages of lung injuries, so we selected the intersection genes of the low-dose group and the high-dose group, which were then used for GO analysis and KEGG pathway analysis. According to GO analysis, approximately 1/4 of the GO terms exhibited an association with oxidative stress, such as oxidation-reduction process, oxidoreduction coenzyme metabolic process, oxidoreductase activity and so on. Interestingly, we also found clear links between KEGG pathways and oxidative stress. For example, ferroptosis, as a unique cell death mechanism, was caused by unrestricted lipid peroxidation ,32. It wtoxicity , it coulAs one type of DNA-binding protein, TFs are considered the control panel of the genome. TFs play the important role in transcriptional regulation and directly shape organism phenotypes . In the Considering the process of lung injury caused by nanoplastics is dynamic and complex, it is of great necessity to add multiple experimental models for comprehensive analysis. Besides the DEGs retrieved from our experiments in vitro, we incorporated the rat lung injury models induced by polystyrene microspheres from the GEO database into this current study. We obtained 16 candidate genes from the microarray analysis of rat lung tissues. After intersecting these findings with the genes of the TF-mRNA regulatory network, we finally identified TNFRSF12A  as the pivotal gene with a consistent trend in our RNA-seq data and the GEO data. Of note, Fn14, the receptor for the Tumor necrosis factor (TNF) superfamily cytokine TWEAK, is well-known for its important role in diverse pathologic processes including oxidative stress, inflammation, angiogenesis, carcinogenesis, proliferation and death ,44,45,46Previous studies on the respiratory toxicity of nanoplastics are far from enough. In this study, we made the following efforts to fill the gaps in this research field. Firstly, instead of a single exposure concentration, we selected different treatment concentrations of PS-NPs in lung cells for RNA-seq simultaneously, allowing us to identify the altered genes with a consistent trend. Secondly, we comprehensively analyzed the underlying mechanisms, and the elevation of ROS was validated with fluorescence. Thirdly, the results of animal experimental data were also included to make our study more reliable. Future studies will employ oxidative stress inhibitors and animal models of lung injury caused by respiratory exposure to nanoplastics to establish the involvement of oxidative stress as a critical molecular event, laying the groundwork for further investigation into nanoplastics-induced inhalation toxicity.Overall, our study revealed the potential respiratory toxicity of nanoplastics. We confirmed that nanoplastics indeed inhibited the viability of human lung epithelial cells mediated by oxidative stress. Through RNA-seq and bioinformatics analysis, we screened nine key TFs that regulated nanoplastic-induced oxidative stress in BEAS-2B cells and constructed the regulatory network. Combined with a GEO database analysis, we identified that FN14, a star molecule involved in the regulation of oxidative stress, may act as an intriguing target in lung injuries caused by nanoplastics.


To reveal their underlying connections, many image-based computational fluid dynamics (CFD) studies have been conducted. However, the methodological validations using both in vivo 4D Flow MRI, in silico CFD, in vitro stereoscopic and tomographic particle imaging velocimetry (Stereo-PIV and Tomo-PIV) techniques. PIV experiments and CFD were conducted under steady state corresponding to the peak systole of 4D Flow MRI.In the present study, we performed a comparative assessment on the hemodynamics of a patient-specific intracranial saccular aneurysm using The results showed that all modalities provided similar flow features and overall surface distribution of WSS. However, a large variation in the absolute WSS values was found. 4D Flow MRI estimated a 2- to 4-fold lower peak WSS (3.99 Pa) and a 1.6- to 2-fold lower mean WSS (0.94 Pa) than Tomo-PIV, Stereo-PIV, and CFD. Bland-Altman plots of WSS showed that the differences between PIV-/CFD-based WSS and 4D Flow MRI-based WSS increase with higher WSS magnitude. Such proportional trend was absent in the Bland-Altman comparison of velocity where the resolutions of PIV and CFD datasets were matched to 4D Flow MRI. We also found that because of superior resolution in the out-of-plane direction, WSS estimation by Tomo-PIV was higher than Stereo-PIV.Our results indicated that the differences in spatial resolution could be the main contributor to the discrepancies between each modality. The findings of this study suggest that with current techniques, care should be taken when using absolute WSS values to perform a quantitative risk analysis of aneurysm rupture.The online version contains supplementary material available at 10.1007/s13239-021-00583-2. On the other hand, the clinical treatment of an unruptured aneurysm also comes with risks. Therefore, once detected, a rupture risk analysis of the existing aneurysm is important.Intracranial aneurysm is an abnormal localized enlargement of an artery in the cerebral vasculature. It is estimated that between 1 and 5% of the general population are affected by this condition and that 20-30% of the affected population have multiple aneurysms.,8 To reveal the underlying connection between hemodynamics and the progression of intracranial aneurysms, various parameters have been studied in the literature. Among them, wall shear stress (WSS) has attracted extensive attention. The accurate calculation of WSS requires three-dimensional three-components (3D3C) velocity data with high spatiotemporal resolutions. Current non-invasive medical techniques such as phase-contrast magnetic resonance imaging (PC-MRI) can provide in vivo insights into the local blood flow and WSS distribution.,27,34 However, the accuracy of WSS in vivo estimation can be limited by imaging noise, artefacts, and relatively low spatiotemporal resolutions available—especially in smaller blood vessels commonly encountered in the brain vasculature.Hemodynamics can potentially help with the risk analysis of aneurysm rupture.,3,39,20 Especially with the advancement of medical imaging in recent years, image-based CFD can be applied in patient-specific studies, making it a potential tool for assisting clinical decision-making in the future. However, uncertainties due to imposed modelling assumptions and variations of solution strategies in CFD produced controversial reports, such as the aneurysm rupture is caused by either high or low WSS.,38,10,23 This is one of the reasons that hinder the clinical translational value of hemodynamics. Hence, the validation of CFD results through either in vitro or in vivo measurements is necessary. Nevertheless, limited computer simulations results, particularly the CFD-based WSS, have been experimentally verified. Optical imaging measurement techniques, such as particle image velocimetry (PIV), are often used for in vitro validations because they provide well-controlled, high-resolution flow fields. The challenge is that the three-dimensional WSS requires a complete velocity gradient tensor, which did not become achievable in optical experiments until the last decade followed by the developments of the full 3D measurement techniques, such as multi-plane stereoscopic PIV (Stereo-PIV), tomographic PIV (Tomo-PIV) and Shake-the-Box (STB).,40,13,4Over the past decades, computational fluid dynamics (CFD) has been extensively applied in various arterial flows due to their ability to simulate instantaneous 3D velocity field and corresponding WSS.in vivo, in vitro, and in silico datasets) is one of the recommended approaches for validating numerical results and providing comprehensive hemodynamic assessments. To date, only one multi-modality study has performed a comparison of WSS obtained by in vivo 4D Flow MRI, CFD, and in vitro STB techniques under pulsatile condition. However, the high cycle-to-cycle flow variations under pulsatile flow can cause large differences in flow measurements between different modalities, which will amplify the variations in velocity-derived parameters. To further bridge this gap and exclude the cycle-to-cycle hemodynamic variations, we investigated hemodynamics in a patient-specific intracranial aneurysm under steady flow conditions using a multi-modality approach. We compared the steady flow results of PIV  and CFD to the peak systole measurement of in vivo 4D Flow MRI. The geometry for PIV and CFD models was based on the 4D Flow MRI velocity field. Steady-state PIV and CFD studies were performed with inlet flow based on in vivo 4D Flow MRI at peak systole. In this study, we presented the similarities and differences in velocity field, vortex, and WSS distributions obtained by 4D Flow MRI, Stereo-PIV, Tomo-PIV, and CFD.In the current active research area of fluid mechanics in biomedical applications, there is a shift of focus from single- to multi-modality studies. Integrating data . From now on, we will refer to the in vivo 4D Flow MRI data as MRI for short.The patient-specific intracranial aneurysm Fig. a is locaBefore being applied to PIV and CFD studies, the segmented raw surface was processed using Vascular Modelling Toolkit (VMTK). Firstly, we applied Taubin smoothing with passbands of 0.45 and 100 iterations. Afterward, the geometry was clipped at the inlet and outlet of the aneurysm to open the inlet/outlet. Flow extensions and caps were subsequently added to the inlet and the outlet. At this stage, the resulting surface (STL)  was imposed. The working fluid in PIV studies is a mixture of water-glycerol with a measured density  of ρ = 1147 kg/m3 and dynamic viscosity of µ = 0.008113 Pa s at 25 °C. The water-glycerol mixture recipe was chosen to reduce the optical distortion at the interface of working fluid and PDMS phantom. By changing the water/glycerol concentration, the refractive index  was matched with that of the PDMS phantom to achieve no visible refraction.The experiment was conducted under the steady flow condition, which corresponded to the averaged peak systole of 9 cardiac cycles measured by 4D Flow MRI Re = 350), respectively.The flow circulation in the aneurysm was driven by a diaphragm fluid pump  Fig. . A therm3 was seeded in the flow. In Tomo-PIV, the flow field was seeded with a diameter of 25–50 µm and density of 1100 kg/m3 fluorescent Rhodamine-B coated PMMA particles. A volume of approximately 48×70×50 mm3 was illuminated by a double pulsed Nd:YLF laser  with the applied laser energy of 27 mJ. A wavelength cut-off filter was equipped on the camera lens to block the laser light. The aneurysm phantom was placed in Plexiglas tanks containing a water-glycerol mixture with the same refractive index of the phantom . In Stereo-PIV, fluorescent dyed polystyrene particles with a diameter of 25 µm and density of 1100 kg/m Before the volume self-calibration, the image pre-processing of subtracting an average time filter was applied to exclude any tracing particles which stick to the wall. For stereo-PIV, additional self-calibration was also performed to correct misalignment between the calibration plate and the laser sheet.Camera calibrations were performed with a two-level calibration plate placed inside the tank filled with refractive index-matched working fluid. For Tomo-PIV, calibration images at 7 positions equally spaced over 35 mm were taken and used for volumetric calibration along the z-direction. For Stereo-PIV, calibration was done for each measurement plane. A third-order polynomial fitting method was applied to map the 3D world position to the camera sensor plane. This yielded an error of approximately 0.2-0.4 pixel for all views in Tomo-PIV. The volume self-calibration, based on the particle images taken for velocity field calculations, reduced the calibration error to 0.1 pixel, which was necessary for accurate Tomo-PIV reconstruction.2. In the Tomo-PIV analysis, the 3D correlation were performed iteratively with 6 steps and with 2 passes in each step. It started with window size 96 × 96 × 96 voxel (75% overlap) and then deformed to a 2 times smaller window size in the following each time step. The final interrogation window size was 16 × 16 × 16 voxel with 75% overlap, resulting in a spatial resolution of 0.25 mm3. All vector fields were post-processed by a median filter (5 × 5) for the outlier detection, removal, and replacement. In addition, parts with a correlation value of < 0.8 were removed. The removed parts were filled up by averaging 100 vector fields. The PIV processing was performed using DAVIS 10.0.5. The 2D velocity results of Stereo-PIV were extended to 3D by interpolating the velocity vectors on 50 planes into a rectangular field.Before cross-correlation, manual geometric masks were created in all particle images to remove the non-flow regions. Due to the Gaussian laser illumination shape, the masked images were then pre-processed firstly with an intensity normalization filter, subsequently with Gaussian smoothing (3 × 3) and sharpening. In the Stereo-PIV analysis, the cross-correlation started with 2 passes at window size 48 × 48 pixel with 50 % overlap and rectangular weight. Then it was followed by 3 passes decreased window size 12 × 12 pixel with 75% overlap using a circular weight. The resulting final spatial resolution was 0.144 mm3 and a dynamic viscosity of 0.0035 Pa s. A rigid wall with no-slip conditions was assumed at the aneurysm wall. The steady flow simulation was performed by solving the incompressible 3D Navier-Stokes equations with the solver settings in Table The CFD simulations were carried out with the finite-volume code ANSYS Fluent 17.1 . The STL geometry was meshed using the ICEM CFD . To properly resolve the boundary layer, the mesh in the proximity of the wall was composed of 12 layers of flat polyhedral elements, with a first-layer thickness of 0.05 mm and an exponential growth rate of 1.2. In the central part of the domain, polyhedral elements were applied. In total, the mesh contained 0.8 million polyhedral cells. The mean WSS difference between this and a finer mesh of 2.25 million cells simulations was less than 1% confirming that our results are sufficient to obtain grid-independent solutions. Blood was modelled as a Newtonian fluid with a density of 1060 kg/m3. The unit inward normal vectors at each surface point were calculated. The velocity gradients obtained by each modality were mapped from the measured fields to the aneurysm surface by inversed-distance weighted interpolation. The value at each surface point was weighted by a function of the distance between each measured data point to the surface data point. The WSS vectors were computed as:WSS was estimated by mapping the velocity gradient from the measured/simulated data to the STL surface grids. As mentioned previously, the STL surface was based on MRI velocity field with a resolution of 0.47 × 0.47 × 0.5 mmwhere i, j = [1–3] are for the 3 Cartesian (x-y-z) coordinates. In the three-dimensional coordinate system, the WSS vectors were calculated as:where indices -, y- and z-direction, while where ref) during comparison of the flow field.In PIV experiments, the aneurysm size was scaled up and a matched Reynolds number was imposed at the inlet, which ensured similar flow patterns to MRI flow. Consequently, the inflow velocity varied from that of MRI. In order to exclude the impact of varied inflow velocity across modalities, we normalized the velocity with the maximum velocity of the inlet  captures nicely the jet inflow and gives a similar shape for all modalities. Almost identical results were achieved between the Tomo-PIV and CFD. The MRI shows a slightly smaller recirculation angle of the jet inflow. This is commonly found in MRI acquisition due to displacement artefacts.The detailed comparisons of the measured and predicted velocity fields were performed next. The iso-surface of normalized velocity magnitude is shown in Fig. Two characteristic cross-sections along the z-coordinate direction  as the vortex detection methods were analysed next. To make the out-of-plane vorticity comparable, we scaled the vorticity level by the average velocity and the vessel size at the inlet:To further evaluate the ability of different modalities in capturing the 3D flow, the vorticity and Q-criterion iso-surface and Fig. Fig. As mentioned previously, there was a variation of fluid viscosity, inflow velocity, and vessel size between PIV experiments and MRI/CFD. To compare WSS quantitatively, we scaled the WSS level by:Note that the same method described in section 2.5 and the original spatial resolution of each modality were used for the WSS calculation. For qualitative comparison, the WSS magnitude was normalized by the WSS at the parent vessel of each modality. Figure  The PIV techniques also produce lower values than CFD does, but the differences are less significant. Figure in vivo MRI. The mean difference is 1.27 Pa, 0.96 Pa, 2.11 Pa for Tomo-PIV, Stereo-PIV, and CFD, respectively. The 95% limits of agreement is ± 3.80 Pa, ± 3.64 Pa, and ± 5.79 Pa for Tomo-PIV, Stereo-PIV, and CFD, respectively. For all modalities, the difference increases when the WSS magnitude is higher.Table in vitro because it can measure the velocity in a volumetric and high-resolution manner.,22 Given that each modality has its strong and weak points, combining data obtained from different modalities has been proposed in order to ensure the reliability of the results and to help the realization of hemodynamic analysis in clinical decision-making. In this work, we conducted a multi-modality study based on a patient-specific intracranial aneurysm. The velocity field, vorticity, and WSS magnitude were compared between Tomo-PIV, Stereo-PIV, in vivo 4D Flow MRI, and CFD. The results reveal that all modalities can capture the flow characteristic of a high-velocity inflow jet and its accompanying vortex ring structure which recirculates the flow towards the upper part of the aneurysm. MRI shows a slightly smaller recirculation angle compared to other techniques. This is commonly found in MRI acquisition due to displacement artefacts. Regarding the velocity field and vorticity, a good agreement was obtained between CFD and PIV measurements . Thus, the averaging effect is severe with low-resolution in the depth direction. It was reported that MRI with a voxel size of 1 mm underestimated WSS by 40% in a noise-free numerical simulation of parabolic flow.,21 Only a few comparative experiments studied the impact of fluid-structure interactions on hemodynamics. These experiments were based on rigid and compliant straight vessels and utilized the 2D PIV technique.,17 Further experimental data based on patient-specific geometries, non-Newtonian fluid solution, and 3D techniques are required to validate the simulation models. In the current study, the inflow boundary conditions in PIV and CFD are steady, which varies from in vivo MRI flow condition. However, we also performed unsteady-state PIV experiments and CFD simulations by imposing a patient-specific waveform at the inlet (see results presented in the Online Resource). The results show that the velocity and WSS distribution at the peak systole are similar to the steady-state. The difference in time-averaged WSS between steady and pulsatile flow condition was studied in Ref. in vitro and in silico studies closer to the real in vivo cases.Several assumptions made in the current PIV and CFD studies, namely the Newtonian viscosity of blood and rigid vessel wall assumptions, could also be associated with the discrepancies. Some numerical studies have reported that neglecting the Non-Newtonian effect of blood and fluid-structure interactions can lead to overestimation of WSS magnitude.in vivo 4D Flow MRI, in vitro PIV (Stereo-PIV and Tomo-PIV), and in silico CFD. Our results demonstrated a good agreement in the flow pattern, velocity, and vorticity between PIV and CFD modalities. However, MRI-based velocity is smaller than velocity based on other techniques in both fast and slow flow areas. The comparison of down-sampled PIV and CFD data to MRI resolution indicates that spatial resolution is the main contributor to the discrepancy. Qualitative agreement in WSS was found across all modalities, but there is a large variation in the absolute WSS values. We observed that the MRI-based WSS magnitude is significantly lower than those based on PIV and CFD. We also found that with a higher out-of-plane spatial resolution, Tomo-PIV gave a higher WSS and better velocity agreement with CFD than Stereo-PIV did. This confirms that spatial resolution plays an important role in the underestimation of WSS. However, the impacts of non-Newtonian viscosity and the compliant wall on WSS should be assessed through in vitro experiments in future studies.We have performed a comparison study of flow and WSS in a patient-specific intracranial aneurysm through Supplementary file1 (DOCX 2908 KB)Below is the link to the electronic supplementary material.


There was in that year a significant increase in lung-heart toxicity and a significant decline in lung-heart function as measured by left ventricular ejection fraction, forced vital capacity and carbon-monoxide diffusing capacity.A cohort of 123 women with stage I-II breast cancer enrolled in a prospective clinical trial of adjuvant radiotherapy who were followed for over 10 years. Year 2020 was associated with excess mortality. The number of deaths . We were concerned whether the COVID-19 pandemic affected the outcomes. All patients were analyzed as a single cohort. Lung-heart status was reverse-scored as freedom from adverse-events (fAE) on a 1–5 scale. Left ventricular ejection fraction (LVEF) and pulmonary function tests were untransformed. Statistical analyses applied least-square regression to calendar-year aggregated data. The significance of outliers was determined using the Dixon and the Grubbs corrected tests. At 12.0 years median follow-up, 103 patients remained alive; 10-years overall survival was 87.8%. In 2007–2019, 15 patients died, of whom 11 were cancer-related deaths. In 2020, five patients died, none of whom from cancer. fAE and lung-heart function declined gradually over a decade through 2019, but deteriorated markedly in 2020: fAE dipped significantly from 4.6–4.6 to 4.3–4.2; LVEF dipped to 58.4% versus the expected 60.3% ; forced vital capacity dipped to 2.4 L vs. 2.6 L ; carbon-monoxide diffusing capacity dipped to 12.6 mL/min/mmHg vs. 15.2 . In conclusion, excess non-cancer mortality was observed in 2020. Deaths in that year totaled one-third of the deaths in the previous decade, and revealed observable lung-heart deterioration. The COVID-19 pandemic waves are unprecedented devastating disease outbreaks. We all have vivid memories of emergency departments and hospital wards overwhelmed with critical cases. Early on, caregivers were aware that cancer patients represented a particularly vulnerable population . There iSuch is the case with the TomoBreast clinical trial. Early meta-analyses of breast cancer treatment data demonstrated a survival advantage by using adjuvant radiotherapy for breast cancer as part of a conservative strategy . Based oFollow-up of the trial was continuous and unabated except for a disruption in 2020 due to the COVID-19 pandemic. Recent studies have observed disruptions in patient care during the lockdown period of the pandemic, including cancer-related care . Such loThe present study aims to investigate up to and through the COVID-19 pandemic and its impact on mortality and lung-heart injuries in a breast adjuvant radiotherapy patient setting as part of the TomoBreast clinical trial.The study population includes participants in the TomoBreast clinical trial which randomized post-surgery stage I-II breast cancer patients into two groups: adjuvant image-guided radiotherapy vs. adjuvant conventional radiotherapy. The trial’s design, summary, flowchart and cardiopulmonary quality of life related results have been detailed elsewhere . The triPatients’ information was abstracted from individual patient’s electronic medical files and from the Belgian federal eHealth platform ,21,22, aHeart and lung toxicities were recorded as the maximal score in any of the Radiation Therapy Oncology Group (RTOG) late morbidity or any of the Subjective Objective Management Analytic/Late Effects on Normal Tissues (SOMA/LENT) cardiac and pulmonary items. The toxicity grades were coded on an ordinal scale, from 0, i.e., G0, no toxicity, to 4, i.e., G4, worst grade.The patients’ toxicity grades were converted into continuous “freedom from adverse event” (fAE) scores ranging from 1 (worst) to 5 (best) by applying fAE = (5—toxicity grade). Though not strictly necessary, the conversion allows the semantic interpretation of a positive slope  as an indicator of health improvement, and a negative slope  as an indicator of deterioration.A total of 848 echocardiography reports were retrieved, averaging 6.9 per patient at different time points. Left ventricular ejection fraction (LVEF) was measured using Simpson’s biplane method. LVEF was reported in 830 exams and was omitted in 18 exams, 3 of which were estimated from the Teichholz method, 3 from Mitral Deceleration Time, and 1 from Tricuspid annular plane systolic excursion (TAPSE), leaving 11 echocardiographic records with truly missing LVEF. The LVEFs were originally reported as continuous exact numeric values in 615 reports, and as interval values of “50–55” in 1 report, “50–60” in 44 reports, and “>60” in 170 reports. The interval values were converted to a mid-interval value of 52.5 for “50–55”, to 55 ± 2 for “50–60”, and to 65 ± 2 for “>60”, taking into account other echocardiographic measurements specified in the reports. In total, 837 LVEF measurements were available.n = 790 records), forced expiratory volume in the first second , peak expiratory flow , vital capacity , total lung capacity , residual volume , functional residual capacity , airway resistance , specific airway resistance , diffusing capacity for carbon monoxide , and alveolar gas volume . The direct measurements were used, without conversion to a percentage of predicted reference populations.Pulmonary function tests were retrieved from 796 reports, averaging 6.5 per patient at different time points. The measurements abstracted were forced vital capacity  score, at D30, an AE score but no LVEF measurement, and at D40, an LVEF but no AE score. The LVEF at D30 is interpolated from the D20 and D40 LVEFs; the AEs at D20 and D40 cannot be interpolated and remain missing.All clinical toxicity and echocardiography and pulmonary function test data were merged into a single data frame ordered by patient and by contact date, resulting in a full data frame with The study’s general approach is that of a cohort monitoring over the calendar years with the intent of detecting adverse changes . For theThe time variable required for survival analysis is the interval between an observation date and a given time origin, i.e., the follow-up year is embedded in the survival model. Unless all patients in a cohort are accrued on the same date, in which case the follow-up period equates the time elapsed, conventional survival analysis cannot detect the effect of a particular follow-up year. In the present study, we simply tabulated the deaths observed over the calendar years, without significance testing. Mortality in the study refers to the crude number of deaths.For discussion purposes—not as study objectives—the overall survival from the date of randomization to death of any cause, and the breast cancer specific survival, from the date of randomization to the date of death from breast cancer, were computed using the Kaplan-Meier product-limit method .https://zenodo.org/deposit/5919956 (accessed on 30 January 2022) with reserved doi:10.5281/zenodo.5919956.All statistical computations were implemented using R version 4.1.2 . Packagen = 1 death), 2012 (n = 1), 2013 (n = 2), 2014 (n = 1), 2015 (n = 3), 2017 (n = 2), 2018 (n = 1), 2019 (n = 4), 2020 (n = 4), and 2021 . None of the five patients who died in 2020–2021 had any evidence of cancer . The calendar years of death were 2011 . The percentages of patients without heart or lung toxicity dipped markedly to 73.3% and 65.8%, respectively (The incidence of cardiac and pulmonary toxicities in 2020 is highlighted in ectively . From TaReverse modeling the toxicity grade as a continuous “freedom from adverse event” (fAE) score that ranges from 1  to 5 , and pooling the fAE as a single variable shown in  indicateSimilar to the patterns of toxicities, the heart and lung function tests deteriorated over time. The heart LVEF, the lung FVC, and the lung DLCO decreased, while the lung RV increased . The LVEAgainst a decade-long backdrop of gradual functional decline, year 2020 was associated with a peak deterioration in almost all cardiopulmonary indicators . Year 20The pattern of deaths appears to mirror the severe excess mortality that affected the general population early in the COVID-19 epidemic . A studyp = 0.004  and breast cancer specific survival  show that the current study’s population have survival rates comparable to other studies of stage I-II breast cancer . Other tAside from the small number of patients, limitations of this study include its descriptive design. Viral serology was not assessed. The evaluation of patients was preponderantly trial related during the first five years, but thereafter the frequency of contacts and examinations were only loosely related to the trial. These later patient visits depended on the clinical circumstances, on patient symptoms, and on increasing co-morbidities with advancing age. The observed excess lung-heart functional deteriorations might have resulted from a poor-health effect of patients requiring more attention, potentially causing a less-fit bias .The strengths of the study include a build on a hybrid study design, combining a prospective randomized controlled trial on a well-defined population with the comprehensive information generated by an observational perspective. The study also builds on the eHealth data exchange platform, a Belgian public federal institution ,21. The This long follow-up study of a cohort of breast cancer patients found a gradual decline in lung and heart function over a decade, which worsened significantly in 2020. Five non-cancer deaths, without evidence of overt SARS-CoV-2 infection, were observed around that first pandemic year. With 15 deaths observed during the entire decade preceding the pandemic, these 5 pandemic-year deaths represented one third of all the deaths that occurred during the prior ten years. This raises the concern that the deaths in the pandemic-year were premature and might have been linked to a reduction of general medical care at the time. The management of future COVID-19 epidemic waves might need to consider how to avoid a radical reduction in medical contacts with patients. The possibility that a history of nodal irradiation could have affected the outcomes deserves attention.


Logistic regression analysis was employed to calculate the odds ratios of abnormalities in voiding parameters for the obese group, with the non-obese group serving as a reference. (3) Results: The obese group had odds ratios of 5.17  for shortened hours of undisturbed sleep (<302 min) by s-HMSU and 7.65 (1.88–31.1) for nighttime urinary frequency by a questionnaire after adjusting for age and sex. In addition, the obese group had an adjusted odds ratio of 2.27 (0.76–6.78) for decreased maximum bladder capacity (<212 mL) by s-HMSU. (4) Conclusion: the results of the present study suggest that nocturia and shortened hours of undisturbed sleep are signs of obesity.(1) Background: Evidence has accumulated regarding the etiology of lower urinary tract symptoms associated with obesity and metabolic syndrome. Therefore, the present study aimed to identify which subjectively and objectively measured voiding parameters were associated with obesity in a community-based population. (2) Methods: Voiding parameters on a self-administered questionnaire and a digital self-health monitoring system for urine excretion (s-HMSU) were compared between participants with and without obesity, defined as a body mass index ≥ 25 kg/m Lower urinary tract symptoms (LUTS) are a major problem in middle-aged and older populations. Nocturia worsens the quality of daily living because of insomnia and also increases the risk of falls/bone fracture, frailty, pneumonia, and cardiovascular disease ,2,3. TheThe etiology of nocturia is multifactorial and now considered to consist of the following three pathological conditions: overproduction of urine , reduction of bladder capacity, and sleep disorders . As the p < 0.05). p = 0.012), with 51.7% for zero, 34.5% for once, and 6.9% for two or three times in the non-obese group, and 23.3%, 56.7%, and 20.0% in the obese group. Mid-nocturnal insomnia (Q2 in the Quick Inventory Depressive Symptomatology-Japanese (QIDS-J)) was significantly worsened in the obese group than that in the non-obese group, with it defined as either “I wake up more than once a night and stay awake for 20 min or more, frequently more than half a week (score 3)” or “I wake up at least once a night, but I go back to sleep easily (score 2)”  for shortened HUS (<302 min) and 7.65  for nighttime urinary frequency  after adjusting for age and sex, with the non-obese group serving as a reference (p = 0.1724) for nighttime urinary frequency, documented by the 24 h voided volume monitoring system, and 2.27  for decreased MBC (<212 mL).The non-obese and obese groups included 29 and 30 participants, respectively. The age and male-to-female ratio showed no statistically significant differences between the two groups . Clinicaeference . In addiThe results of the current study indicate the following: (1) Self-reported nighttime urinary frequency (Q2 in CLSS) was greater in the obese group than in the non-obese group, while that measured by s-HMSU tended to be higher in the obese group than in the non-obese group. (2) HUS measured by s-HMSU was significantly shorter in the obese group than in the non-obese group. (3) Mid-nocturnal insomnia obtained from the self-reported questionnaire (QIDS-J Q2) was significantly worse in the obese group. These novel findings suggest that nighttime urinary frequency and shorted HUS are closely related to, and may be a notable sign of, obesity.Of the three major causes of nocturia  , nocturnThe HUS is defined as the time from falling asleep to first awakening, which may be another key factor affecting nocturia-related sleep disturbances and quality of life . Taking In Japan, MetS is defined as an abdominal circumference ≥85 cm in men and ≥90 cm in women, accompanied by any two or more of the three disorders  . In the In the present study, MBC in the obese group tended to be lower than that in the non-obese group. The reduced bladder capacity commonly occurs with aging  ,25. In ap < 0.05) [Our KDHP conducted an interventional study using digital health approaches for comprehensive lifestyle modifications among individuals with obesity and/or MetS, which is increasing in Japan . In this < 0.05) . TherefoThis study had several limitations. First, the number of participants in the present study was small, which could have affected a wide range of confidence intervals. Second, the single-day monitoring period of the s-HMSU was insufficient considering that the recommended period for FVC monitoring is ≥ 3 days . Third, 2 [This observational study was conducted as part of the KDHP between June 2017 and December 2019 . Individ2 . After, Participants were asked to fill out a questionnaire including the CLSS and the QIDS-J score . NighttiTo measure the 24 h voided volume, we employed the s-HMSU system  ,14. Cali2). Fasting blood glucose, hemoglobin A1c (HbA1c), serum total cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides, and creatinine levels were measured using standard methods.Other data collected during the health checkup included age, sex, anthropometric indices, blood pressure, and blood chemistry results in the fasting state. Body height and weight were measured and BMI was calculated as weight (kg)/height squared  of each voiding abnormality for obesity, with non-obesity serving as a reference. The model incorporated age and sex as covariates. The cutoff value of NPi in the chi-square test was adopted as 33% because nocturnal polyuria in middle-aged and older people is defined as >33% of NPi [® Pro version 15.0.0 software . All probability values were two-tailed and the significance level was set at p < 0.05.Continuous variables are expressed as mean and standard deviation or median and interquartile range. Basic clinical characteristics and voiding parameters were compared between the obese and non-obese groups using the unpaired % of NPi ,25. In cThis study demonstrated an association between obesity and nocturia in a community-based population. Nocturia and a reduction in HUS could be signs of obesity.


Meningiomas are mainly benign intracranial tumors. Nevertheless, risk of recurrence exists in long-term follow-up, so new prognostic markers are still need to be identified. MIB-1 is no diagnostic criterion in WHO classification of meningiomas by now. This retrospective study shows that MIB-1 as well as mitotic count are good predictors for progression-free survival in skull-base meningiomas. The implantation of MIB-1 may enable an improved classification of meningiomas regarding progression-free survival. Moreover, this analysis of skull-base meningiomas shows that current cut-offs may have to be adjusted for meningioma location.Although meningiomas are mainly non-aggressive and slow-growing tumors, there is a remarkable recurrence rate in a long-term follow-up. Proliferative activity and progression-free survival (PFS) differs significantly among the anatomic location of meningiomas. The aim of the present study was to investigate the predictive power of MIB-1 labeling index and mitotic count (MC) regarding the probability of PFS in the subgroup of skull-base meningiomas. A total of 145 patients were included in this retrospective study. Histopathological examinations and follow-up data were collected. Ideal cut-off values for MIB-1 and MC were ≥4.75 and ≥6.5, respectively. MIB-1 as well as MC were good predictors for PFS in skull-base meningiomas. Time-dependent analysis of MIB-1 and MC in prediction of recurrence of skull-base meningioma showed that their prognostic values were comparable, but different cut-offs for MC should be considered regarding the meningioma’s location. As the achievement of a gross total resection can be more challenging in skull-base meningiomas and second surgery implies a higher risk profile, the recurrence risk could be stratified according to these findings and guide decision-making for follow-ups vs. adjuvant therapies. Although meningiomas—especially World Health Organization (WHO) grade 1 and 2—are mainly non-aggressive and slow-growing tumors, there is a remarkable recurrence rate in a long-term follow-up . There iA total of 880 patients were surgically treated for WHO grade 1 and 2 meningioma between 01/2009 and 07/2019 at our neurosurgical department. Patient data were retrospectively reviewed. Institutional review board approval had been obtained. Histopathologically confirmed meningioma, intracranial localization, age equal or greater than 18 years, the availability of the MIB-1 index and/or MC, and neurosurgical resection were inclusion criteria for this study. Meningiomas outside of skull-base or associated with neurofibromatosis type 2 were excluded due to differences regarding histopathology and proliferation potential ,11. PatiClinical information including age, sex, comorbidities, Karnofsky performance status (KPS), body mass index (BMI), peritumoral edema, tumor growth pattern, WHO grading based on postoperative histopathological examination, immunohistochemical examinations, extent of tumor resection based on the Simpson grading system according to the European Association of Neuro-Oncology (EANO), surgical and medical complications according to the classification of Landriel Ibañez , and posThe following histopathological investigations were previously described ,16,17: HAs institutional routine, clinical and imaging follow-up consisted of MRI scans at 3 months after surgery as well as on an annual basis for the following years . EarlierData were organized and analyzed using SPSS . Descriptive statistics of baseline characteristics included mean and standard deviation (SD) for continuous variables and absolute and relative frequencies for categorical variables. Receiver-operating characteristic (ROC) analysis was performed to evaluate how MIB-1 index and MC discriminate between meningioma recurrence. The selection of cut-off points was based on the Youden index . Kaplan–In all, 145 patients were included in the present study. The mean age was 59.9 years and the female/male ratio was 2.6:1. The mean KPS before surgery was 90.9. Tumor grading according to the WHO classification criteria [p = 0.06) and 0.58 , respectively. Sensitivity and specificity of MIB-1 labeling index at the threshold of 4.75 (according to Youden index) were estimated to 89% and 46%, respectively. For MC, sensitivity and specificity at the threshold of 6.5 were 22% and 98%, respectively. Mean MIB-1 labeling index was 4.9 ± 2.3% and 1.3 ± 2.2 for MC. The AUC of MIB-1 labeling index and MC for intracranial recurrent meningioma were 0.71  and patients with an MIB-1 labeling index of ≥4.75% had a median PFS time of 402 weeks (95% CI: 358–445). Patients with an MC of <6.5% had a median PFS time of 406 weeks (95% CI: 376–436) and patients with an MC of ≥6.5% had a median PFS time of 250 weeks (95% CI: 71–429) .Results of the time-dependent ROC analysis of MIB-1 and MC are shown in Primary treatment of cranial meningioma is a maximum cytoreductive approach, that mostly results to be curative . In certNevertheless, there is still an incongruence between the WHO grade and clinical course, so new prognostic markers are still need to be identified . MIB-1 aMIB-1 stains nuclear protein Ki67 which is expressed in proliferation phases of the cell cycles . It’s beThe static allocation of all meningiomas regardless of their origins—e.g., skull-base, non-skull-base and spinal—appears to be inaccurate as the location has considerable impact on meningioma PFS, especially in skull-base meningiomas ,30. In tIntuitively, MC was associated with meningioma recurrence but with a slightly higher cut-off as in the WHO classification . MIB-1 wAlthough MIB-1 is not a classification criterion by now, many neuropathologists determine MIB-1 already as a routine. This study should not induce to replace MC by MIB-1 but to use it in addition. We suggest that in the modern era of tailored neuro-oncological care a maximum of potential molecular markers may be used to achieve a most efficient and reliable individual treatment as well as follow-up scheduling. The additional assessment of MIB-1 regarding tumor recurrence risk could improve stratification of patients to specific follow-up. Nevertheless, this study also emphasizes that different cut-offs for both, MIB-1 and MC, may be necessary in order to comply with the different pathophysiology depending on meningioma location.MIB-1 as well as MC are good predictors for PFS in skull-base meningiomas. Their prognostic value is more or less equal. We do not want to substitute MC with MIB-1, rather emphasize the possible benefit of MIB-1 as an additional histopathological factor. Moreover, higher cut-off for MC should be applied in predicting PFS among skull-base meningiomas, but further studies are needed.As the achievement of a gross total resection can be more challenging in skull-base meningiomas and second surgery implies a higher risk profile, the recurrence risk could be stratified according to these findings and guide decision-making for follow-ups vs. adjuvant therapies.


The direct targets of microRNA-569 were determined by using bioinformatics analysis and a dual-luciferase reporter assay. The expression level of microRNA-569 was down-regulated in PC patients with a poor prognosis. In vitro and in vivo experiments indicated that over-expression of microRNA-569 inhibited the migration and invasion of PC cells. MicroRNA-569 negatively regulated NUSAP1 by directly binding its 3'-untranslated region. Further mechanism research implied that the ZEB1 pathway was involved in microRNA-569/NUSAP1 mediation of the biological behaviors in PC. These data demonstrated that microRNA-569 may exert a tumor-suppressing effect in PC and maybe a potential therapeutic target for PC patients.MicroRNAs (miRNAs) are known to be involved in the development and progression of pancreatic cancer (PC). In this study, the prognostic significance and mechanistic role of microRNA-569 in PC were explored. Quantitative real-time PCR was used to detect the expression of microRNA-569 in PC tissues and cell lines. Scratch test and Transwell assay were conducted to detect migration and invasion ability. The xenograft nude mice model was used to determine tumor metastasis  Pancreatic cancer is often referred as the “king of cancers”, with a very poor prognosis, and is the fourth leading cause of cancer death in the world . The maiMicroRNAs (miRNAs), containing 22 to 25 nucleotides, belong to single-stranded ribonucleic acid and exert function by degrading or inhibiting the translation of other proteins to regulate gene expression . DysreguNucleolar and spindle-associated protein 1 (NUSAP1), mainly modulates spindle assembly and stability during mitosis . PreviouOur research is the first to investigate the miRNA-569 expression in PC and reveal its relation with clinical outcomes. Furthermore, the biological role of miRNA-569 and its potential molecular mechanisms were investigated. miRNA-569 directly targets NUSAP1, which in turn regulates ZEB1 expression and inhibits PC cell migration and invasion.Patients with pancreatic cancer were divided into the following two groups: poor prognosis, 12 months or less; and good prognosis, above 21 months according to the survival time. As shown in PC is prone to metastasis in the late stage, especially liver metastasis, leading to a poor prognosis. The expression levels of miR-569 after transfection of mimics in SW1990 and Capan-2 cells were confirmed. As expected, miR-569 levels were significantly up-regulated after transfection of mimics . Scratchin vivo. Capan-2 was transfected with agomir-569 and control, respectively. Transfected cells were injected into the spleen of mice to establish the liver metastasis model, and the metastatic focus was evaluated ten weeks later  and SW1990(#TCHu201) were acquired from Suer Biological Technology  and the Type Culture Collection of the Chinese Academy of Sciences , respectively. Both the cell lines were cultured in DMEM containing 10% FBS, 100 units/ml penicillin-streptomycin at 37° C containing 5% CO2. PC tissue specimens were gathered from the Department of Pathology, the Affiliated Shengjing Hospital, China Medical University, and confirmed by histopathological examination by two pathologists. More detailed patient information has been described in the earlier paper .Total RNA of cultured cells was extracted with Trizol reagent according to the manufacturer’s instructions, and the RNA was stored at -80° C. The concentration and purity of RNA were measured (RNA purity =A260/A280), and the One Step PrimeScript® miRNA cDNA Synthesis kit was used for reverse transcription (RT). Quantitative real-time PCR was performed on ABI 7500 Real-time PCR system (Applied Biosystems) using SYBR Premix Ex Taq II. All the reactions were performed for triplicates. Primer sequences were listed in in vitro using jetPRIME reagent (Polyplus) according to protocol.NUSAP1 siRNA was designed and synthesized by GENEWIZ . The NUSAP1 plasmid was purchased from GeneChem . miRNA-569 mimics and its control were synthesized and gained from RiboBio . Cells were transiently transfected The siRNA sequences of NC and NUSAP1 were listed:siNUSAP1-1: 5’-GGAAGACUCUCUGUGGUUTT-3’siNUSAP1-2: 5’-CCAAGACUCCAGCCAGAAATT-3’NC siRNA: 5’-AATTCTGCGAACGAGTCACGT-3’As shown in When the transfected cells reached sufficient confluency, the cell monolayers were scratched across with a 200-ul pipette tip to create a linear wound. Then the supernatant at each well was replaced with a fresh medium without FBS. Migration images were captured at 0, 24 h after scratching by using an inverted bright-field microscope.4 cells was placed in the upper chamber, and 500 μL medium containing 10% fetal bovine serum was added to the lower chamber. As for the invasion assay: the Matrigel was diluted with precooled serum-free medium at a ratio of 1:30, and added 50 μL into the upper chamber. Other procedures were the same as the migration assay. 24 hours later, the chamber was taken out and fixed with 75% ethanol. After staining with Reyes-Giemsa, put it under a fluorescence microscope  for observation and take photos.As for the migration assay: 200 μL serum-free medium containing 3×10Western blot was conducted as previously described . The priDual-luciferase reporter assays were performed as we previously described . Briefly6) were labeled with luciferase ahead of time. After transfection with agomir-NC (5μM) or agomir-569 (5μM), transfected cells were injected into the mouse spleen (n=5 in each group), respectively. 10 weeks after injection, the mice were killed according to the requirements, and the liver tissues were collected and embedded in paraffin. Then HE staining and pathological analysis was performed. The relevant experimental scheme and content were approved by the Ethics Committee of China Medical University .Female BABL/c nude mice  were got from Vitalriver . Capan-2 cells (1×10http://www.targetscan.org/vert_72/) was used to determine the potential miR-mRNA interactions. The predicted target genes are intersected with aberrantly expressed data from the TCGA portal  database displayed by a Venn diagram. GO annotation and KEGG pathway of target genes were performed by DAVID 6.8 software (https://david.ncifcrf.gov/). The results were visualized with the “clusterProfiler _3.11.0” package of the R 3.6.0 language. PPI networks were generated by STRING 11.0 (http://string-db.org), which aimed to assess and integrate proteins from prediction or experiments. The interaction network was visualized by Cytoscape 3.8.1 and the MCODE plugin was conducted to screen potential clusters. In the process of selecting model parameters, the minimum absolute contraction and LASSO regression methods are widely used. The “glmnet 4.1.2” package was selected for modeling research. The Oncomine database (http://www.oncomine.org) was applied to analyze the transcription expression level of the NUSAP1.The public database TargetScan . All experiments were carried out in triplicate. Group comparison was performed by Student’s t-test and the threshold of significant difference was p<0.05.Supplementary Figure 1


In this work, a novel and stable zwitterionic polymer (TPE-CB PUs) was prepared to realize a cellular imaging system. TPE was conjugated into the backbone of zwitterionic polyurethane, which could be well dispersed in aqueous solution and emitted strong blue fluorescence because the TPE segment was aggregated in the core of TPE-CB PUs micelles. More importantly, the TPE-CB PUs micelles showed significant stability in a large pH window and with different storage times. In addition, the micelles exhibited low cytotoxicity in HeLa cells and mainly distributed in the cytoplasm after being incubated with cells. The outstanding properties of TPE-CB PUs combining the merits of AIE and a zwitterionic segment highlight its potential for use as a cell imaging material with remarkable capability. In this work, a novel and stable zwitterionic polymer (TPE-CB PUs) was prepared to realize a cellular imaging system. Moreover, inorganic materials such as quantum dots usually have high cell toxicity, which greatly limits their biological applications. Therefore, development of fluorescent materials with good biocompatibility and a high signal-to-background ratio is urgently needed. Recently, a new class of fluorescent dyes with aggregation-induced emission (AIE) characteristics has been extensively used for the design of cellular imaging fluorescent probes.9–14 AIE behavior has attracted great attention because it can effectively avoid the ACQ encountered in conventional dyes.15–20 Furthermore, AIE fluorogen only light up under aggregate conditions, which give rise to exceptionally high signal-to-background ratio. Among them, tetraphenylethylene (TPE) is a typical AIE molecule.21–28 Once aggregated, the restriction of intermolecular rotation diminishes the non-radiative decay of the excited state energy, leading to aggregates with high quantum yields. More importantly, the TPE is very easily to modify, which make it possible to design probes based TPE for imaging of cells.With the development of fluorescence microscopy techniques, cell imaging has been widely used to study cell biology and biological systems.15–18,21,29,30 However, AIE probes with better biocompatibility, biodegradability and stability is necessary for biomedical applications. Amphipathic polymer, which could self-assembly into core–shell micelles, is a promising strategy for delivering AIE molecule. In our previous study, we have synthesized polyethylene glycol (PEG) conjugated TPE and can form nanoparticles in aqueous solution, which showed low the critical micelle concentration (CMC), low cytotoxicity and photostability.17 PEG is the most popular antifouling materials used for biomedical application. However, it has been reported that the PEG can be oxidized by alcohol dehydrogenase, leading to lose its non-fouling capability in biological environment. Recently, zwitterionic polymers have attracted considerable attention as the alternative of PEG due to its better antifouling ability to prevent protein adsorption.23,31–44 Jiang's group39,45–47 has prepared many kinds of surfaces coated with PCBMA, PSBMA or PMPC, all the results exhibited that these surfaces have excellent antifouling properties and stability in vivo.Until now, many kinds of TPE derivatives were utilized for cell imaging.48,49 Moreover, PU is a well-established biodegradable polymer and its biocompatibility makes it suitable for application in vivo.However, most of the zwitterionic materials prepared using zwitterionic methacrylate through free radical polymerization or living polymerization. Unfortunately, the backbone of these polymers was non-biodegradable and the materials could not be eliminated through metabolism. It compels us to pursue for new method to synthesize zwitterionic polymers. Polyurethane (PU) is known to be one of the most versatile polymers. The structure and properties of PU can be tailored by varying the constituents of these hard and soft domains.via polyaddition reaction to construct an amphiphilic copolymer with AIE fluorescence. As shown in In this contribution, zwitterionic monomer , hexamethylene diisocyanate (HDI) and TPE-based monomer  were condensed 4, tert-butyl acrylate and KI, K2CO3 were acquired from Aladdin. Other solvents and reagents all came from commercial sources and were used as received without further purification. Deionized water was used in all process of the experiments.Hexamethylene diisocyanate (HDI), diethanolamine, iodomethane, 2-bromoethanol, trifluoroacetic acid (TFA) were purchased from Adamas. Zinc powder, 4-hydroxybenzophenone, TiCl1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker AV 400 spectrometers  with deuterated solvents . Mass spectrometry was used to confirm the molecular weight. UV-visible absorption spectra were measured by Analytic-jena Specord S600 (Germany) spectrophotometer in absorbance mode using quartz cuvettes of 1 cm path length. Fourier transform infrared spectroscopy was detected by a Shimadzu Spectrum 8400 spectrometer (Japan) in diffuse reflectance mode. GPC analyses of polymers were performed by HLC-8320GPC and using DMF as the eluent. Perkin Elmer LS-55 spectrofluorometer was used to measure the fluorescence spectra. The excitation wavelength was set at 350 nm, with a slit width of 10 nm. The size distribution of the micelles in water was determined by Zetasizer Nano S90 from Malvern Instruments equipped with a He–Ne laser at a wavelength of 633 nm at 25 °C. The samples were filtered by a 0.45 μm millipore before measurement. To amplify the micelles, TEM images were conducted on a JEM-1200EX microscope operated at 100 kV, the TEM specimens were made by placing a drop of the micelles solution on a carbon-coated copper grid.tert-butyl acrylate  were dissolved in a flask with methanol (35 mL). After 48 hours' reaction at room temperature, the methanol was removed under reduced pressure to obtain the crude product, and then the crude product was purified by silica gel chromatography with dichloromethane (DCM) and methanol (MeOH) as eluent  and a colorless oil (A) was obtained.Diethanolamine  and N-Propionate-diethanolamine  and iodomethane  were dissolved in a 100 mL flask with acetonitrile (35 mL). The reaction was held at room temperature for 48 hours, the acetonitrile was then removed under reduced pressure to obtain the product B (yellow oil). Its chemical structure was confirmed by 1H NMR in methanol-D4 on a 400 MHz spectrometer.This produce B was added into a solution  and stirred vigorously for 3 hours, the mixture was added into anhydrous ether drop by drop for two times and a yellow oil product C was obtained from vacuum drying  were dissolved in a 250 mL flask with a reflux condenser at 110 °C for 24 hours. After cooling the mixture to room temperature, 150 mL deionized water was added and extracted with DCM for five times. The crude product was purified by a silica gel column using a mixture of petroleum ether : ethyl acetate  and a white crystalline solid (D) was obtained. In the second step, D , zinc powder  and 300 mL THF were added into a three-necked flask equipped with a magnetic stirrer at 0 °C with an N2 atmosphere. Then, TiCl4  was slowly added with the temperature under 5 °C. The mixture stirred at the room temperature for overnight. After this, 200 mL hydrochloric acid (1 mmol L−1) was added and extracted with DCM for three times. The crude product was purified by a silica gel column and evaporated under vacuum to get product (E) as a fallow solid. The obtained fallow solid was HO-TPE-OH and its chemical structure was confirmed by 1H NMR in CDCl3 on a 400 MHz spectrometer and mass spectrometry.To synthesize HO-TPE-OH Scheme S2, first i1H NMR and infrared spectrum were used to confirm its chemical structure.The TPE-based polyurethane was synthesized according to a standard procedure. Briefly, amount 0.5 g DHCB was placed in a 50 mL round-bottom flask with a stir bar at 105 °C for 2 hours to remove any trace of water in the system through reduced pressure. And then cooled to room temperature, 0.897 g HDI was added into the flask, and 5 mL DMF was added to dissolve them, then 0.0002 g catalyst dibutyltin dilaurate was injected sequentially. Temperature was then raised to 85 °C, after 4 hours of polymerization under a nitrogen atmosphere, 0.5 g HO-TPE-OH was added to the mixture as the chain extender, after that 2 mL methanol was added to terminate with the residual isocyanate group. The reaction mixture was precipitated in cold anhydrous ether. Fallow polyurethane (TPE-CB PUs) was collected through filtration followed by drying under vacuum. TPE-CB PUs micelles were prepared by a dialysis method. In brief, TPE-CB PUs (10 mg) was dissolved in a mixture of methanol (2 mL) and DMSO (2 mL). Afterwards, 10 mL deionized water was dropwise added under vigorous agitation, continued for 3 hours. The generated solution was transferred into a dialysis bag (MWCO = 3500 Da) and dialyzed against deionized water for two days to remove any residual solvent. Subsequently, the solution was filtered through 0.45 μm pore-sized microporous membrane.Dimensions of micelles were determined by DLS. A Malvern Z90 Zetasizer equipped with a 633 nm He–Ne laser and an avalanche photodiode detector was provided to characterize the hydrodynamic size of the self-assemblies. With the room temperature, the scattering light at an angle of 90° was detected and used to analyse the size and distribution. Additionally, the morphology of TPE-CB PUs micelles was observed by TEM.5 cells per mL in 100 μL media containing 10% FBS, respectively. After cells attachment for 24 hours, the cells were cultured with 10, 50, 100, 200, 500 μg mL−1 TPE-CB PUs micelles and HO-TPE-OH solution for 48 hours. Then PBS was used to wash all samples for a few times to remove the free materials. 10 μL of CCK-8 dye and 100 μL of DMEM cell culture medium were added to each well and incubated for 4 hours at 37 °C. Plates were then analysed with a microplate reader (Thermo Multiskan GO). The formazan dye absorbance was measured at 450 nm, with the reference wavelength at 600 nm. The number of live cells was proportional to the values. The percent reduction of CCK-8 dye was compared to controls (cells not exposure to TPE-CB PUs and HO-TPE-OH), which represented 100% CCK-8 reduction. Three replicate wells were used per microplate, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD).The cell viability of TPE-CB PUs micelles and HO-TPE-OH solution on HeLa cells was evaluated by cell counting kit-8 (CCK-8) assay, respectively. Briefly, cells were seeded in 96-well microplates at a density of 1.5 × 102 in culture medium. Culture medium was changed every 3 days to maintain the exponential growth of the cells. A day before treatment, cells were seeded in 6-well microplates with a density of 1 × 105 cells per dish. Then, the cells were incubated with TPE-CB PUs micelles at a certain concentration of 50 μg mL−1 for 3 hours at 37 °C. Afterward, the cells were washed three times with PBS to remove the TPE-CB PUs micelles and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were taken with a confocal laser scanning microscope (CLSM) Zeiss 710 3-channel  with the excitation wavelength of 350 nm.HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 1% penicillin, and 1% of streptomycin. Cell culture was maintained at 37 °C in a humidified condition of 95% air and 5% COTPE is an iconic AIE fluorogen: it is fluorescence free in good solvents, but becomes emissive in a poor solvent. The simple structure makes it an ideal model for construction of various fluorescent materials. Therefore, TPE-based polyurethane micelles with zwitterionic shell possess great potential for various bio-applications. In this article, TPE-CB PUs was synthesized by a simple polymerization. The synthesis routes were shown in 1H NMR spectra of HO-TPE-OH were illustrated in Fig. S2.1H NMR spectra were marked and conformed well to the molecular structure. The mass spectra were further confirmed the structure of HO-TPE-OH  of 1.86.As shown in TPE-CB PUs contains hydrophobic segments (HO-TPE-OH) and hydrophilic segments (DHCB). Therefore, the micelles could be easily formed in the aqueous solution with hydrophilic DHCB as the zwitterionic shell. While, the hydrophobic TPE was encapsulated in the core, leading to the enhanced quantum yield of the micelles. To confirm the TPE-CB PUs could self-assemble into micelles in aqueous solution, the size of the TPE-CB PUs was measured by DLS. As shown in The morphology of the micelles was further investigated by TEM , which s17,22 In addition, as observed in the inset of The fluorescence property is one of the important parameters to confirm whether the TPE-CB PUs micelles could be used in cell imaging. As displayed in the −1, The CMC value , which have great potential in the biomaterials. Based on the cells viability results, TPE-CB PUs are considered to be promising candidates for cell imaging applications due to their biocompatibility and fluorescence properties.Biocompatibility evaluation is an indispensable step for biomedical applications of materials. The cytotoxicity of material was analysed by the CCK-8 assay. In this work, HeLa cells were chosen as the models for cell tracking. As shown in To explore the TPE-CB PUs micelles for bio-imaging application, HeLa cells was incubated with the micelles for 4 hours and observed by confocal laser scanning microscopy (CLSM). As shown in In summary, a novel and stable zwitterionic polymer with TPE was designed to realize a cellular imaging system. TPE was conjugated into the backbone of polyurethane, which could be easily self-assemble into micelles and display an excellent fluorescence in the aqueous solution. They can be well dispersed in aqueous solution and emitted strong blue fluorescence due to the TPE segment was aggregated in the core of TPE-CB PUs micelles. More importantly, the TPE-CB PUs micelles showed significant stability in a large pH window and with different storage times. Thus, the micelles showed a great potential in bio-imaging and physiological applications. In addition, the micelles exhibited low cytotoxicity in HeLa cells and mainly distributed in the cytoplasm after incubated with cells. The outstanding properties of TPE-CB PUs combined the merits of AIE and zwitterionic segment highlight its potential to use as a remarkable capability of cell imaging materials.There are no conflicts to declare.RA-008-C7RA13238G-s001